The invention described herein was made in course of, or under U.S. Department of Energy Grant DE-FO-G2-85 and ER-60295 and a NIH Cancer Research Training Grant 05137-10.
The major thrust of the prior art concerning the use of radiolabeled monoclonal antibodies (Mabs) is focused on the development of their applications in imaging and therapy. Evidence suggests that Tc-99m possesses ideal characteristics for imaging and Re-186 or Re-188 for therapy. However, the procedures for labeling Mabs with these radionuclides remain inefficient and cumbersome. Unless the labeling processes are made simple, efficient and reliable, the use of Mabs for diagnostic imaging will remain restricted.
Technetium-99m possesses the attractive characteristics of convenience, inexpensiveness, low radiation dose, and suitability for planar and single photon emission computerized tomography (SPECT) imaging. There has been a continuous development of technology to incorporate Tc-99m in all molecules intended for diagnostic imaging applications. Monoclonal antibodies are no exception. Since Tc-99m has only a 6 hour half-life, in most cases the biological half-life of Tc-99m labeled antibody will be determined by the half- life of Tc-99m and not by the clearance time of the IgM, IgG or fragmented antibody. It is for these reasons that TC-99m continues to be the favorite radionuclide for most imaging applications of Mabs.
Indirect labeling of Mabs with Tc-99m via bifunctional chelating agents (BFCA) such as DTPA, cyclam, and diaminodithio (N.sub.2 S.sub.2) compounds is well known in the art. A number of investigators have labeled antibodies with Tc-99m via the use of BFCAs. Khaw, B.A., Strauss, W. and Carvalho, A. et al., Tc-99m Labeling of Antibodies to Cardiac Myosin Fab and to Human Fibrinogen, J. Nucl. Med. 23, 1011-109, 1982; Paik, C.H., Pham, L.N.B., Hong, J.J. et al., The Labeling of High Affinity Sites of Antibodies With Tc-99m, Int. J. Nucl. Med. and Biol. 12, 3-8, 1985; Thakur, M.L., Richard, M.D., and White, F.W., III, Monoclonal Antibodies as Agents For Selective Radiolabeling of Human Neutrophils, J. Nucl. Med. 29, 1817-1825, 1988; Franz, J., Volkert, W.A., Barefield, E.K., et al. The Production of Tc-99m Labeled Conjugated Antibodies Using a Cyclam-Based Bifunctional Chelating Agent, Int. J. Nucl. Med. Biol. 14, 569-572, 1987; and Fritzber, A.R., Kasina, S., Reno, J.M. et al., Radiolabeling Antibodies Using N.sub.2 S.sub.2 Ligands, J. Nucl. Med. 27, 957-958, 1986. Perhaps the most prominent method among them uses the diaminodithio compound as a BFCA. However, even this method requires a lengthy and cumbersome procedure that has prompted researchers to investigate direct labeling methods.
Direct labeling methods offer the advantages of 1) providing higher specific activity (mCi/mg); 2) eliminating the need for conjugation with a bifunctional chelating agent; 3) being less likely to alter the immunospecificity of the Mab; 4) being more likely to be adaptable to an instant "kit" labeling technique; and 5) being useful for labeling antibodies with radionuclides such as Re-186 or Re-188 for therapy.
Human serum albumin has been labeled with Tc-99m by the direct tin reduction method since 1971. The method was published by Lin et al., Use of Fe(II) or Sn(II) Alone for Technetium Labeling of Albumin, Journal of Nuclear Medicine, Vol. 12, No. 5, pp. 204-10, and modified by Eckelman et al., .sup.99m Tc-Human Serum Albumin, Journal of Nuclear Medicine, Vol. 12, No. 11, pp. 707-710 (1971). The method was adapted for labeling antibodies by Pettit Improved Protein Labeling By Stannous Tartrate Reduction of Pertechnetate, Journal of Nuclear Medicine, Vol. 21, No. 1, pp. 59-62 (1980); Huang et al. Detection of Bacterial Endocarditis with Technetium-99m-Labeled Antistaphylococcal Antibody, Journal of Nuclear Medicine, Vol. 21, No. 8, pp. 783-786 (1980); and Rhodes et al., Technetium-99m labeling of Murine Monoclonal Antibody Fragments, Journal of Nuclear Medicine, Vol. 27, No. 5, pp. 685-693 (1986). At least in one case (Rhodes) the resulting labeled product has been reported to be unstable, requiring an elaborate system for purification of the antibody. Sundrehagen et al., Formation of .sup.99m Tc-Immunoglobulin G Complexes Free from Radiocolloids, Quality Controlled by Radioimmunoelectrophoresis, European Journal of Nuclear Medicine, Vol. 7, pp. 549-552 (Spring 1982), appreciating that the tin used in all these methods leads to the formation of Tc-99m-Sn-colloid, used a concentrated hydrochloric acid and gentisic acid reduction method for labeling immunoglobulin with Tc-99m. In order to avoid the colloid formation of Tc-99m, Blok et al., A New Method for Protein Labeling With .sup.99m Tc, Nucl. Med. Biol., Vol. 16, No. 1, pp. 11-16 (1989), used a combination of dimethylformamide and 5M HCl in which Tc-99m was heated at 140.degree. C. for 4 hours. The reduced Tc-99m was then extracted in CHCl.sub.3, evaporated to dryness, and incubated with antifibrin antibody for 1 hour at 40.C. However, these methods are lengthy, cumbersome, and are not adaptable to an instant kit procedure.
Stiegman et al., The Importance of the Protein Sulfhydryl Group in HSA Labeling with Technetium-99m, Proceedings of 22nd Annual Meeting, J. Nucl. Med., Vol. 16, No. 6, suggested that the mechanism of Tc-99m binding was related to sulfhydryl groups. There are approximately 70 cysteine amino acid residues per IgG molecule, leading to approximately 175 disulfide bonds in an IgM molecule, approximately 35 in IgG, and approximately 25 in F(ab').sub.2. In the Fab fragment, there may be as many as 12 groups per molecule.
Liang et al., Serum Stability and Non-Specific Binding of Technetium-99m Labeled Diaminodithiol for Protein Labeling, Nucl. Med. Biol., Vol. 14, No. 6, pp. 555-561 (1987), suggested the importance of the helical structure of antibodies for the preservation of the Mab specificity and immunologic activity. However, Rhodes demonstrated that reducing disulfide bonds to sulfhydryl groups not only allowed them to label murine Mab fragments with Tc-99m, but also preserved the immunoreactivity of the protein. This reduction was achieved by incubating 600 .mu.m of antibody with 5mM SnCl.sub.2 for 21 hours. The product was then lyophilized, ready for adding Tc-99m pertechnetate. Presumably, the excess of SnCl.sub.2 reduced Tc-99m for binding to the sulfhydryl groups. Following a 30 minute incubation, the reaction mixture was passed through a purification column designed to eliminate pertechnetate ions and other technetium impurities. Schwartz, A. and Steinstrabber, A., A Novel Approach to Tc-99m Labeled Monoclonal Antibodies, J. Nucl. Med. 28, 721, 1987, considered that an excess of SnCl.sub.2 at pH 6-7, may lead Tc-99m to the formation of colloid. Schwartz et al. achieved the reduction of antibody disulfide groups with 2-mercaptoethanol (ME). Excess reagent was eliminated by molecular gel filtration and the protein was lyophilized. Reduction of pertechnetate was achieved by the use of commercial pharmaceutical kits such as pyrophosphate, which contains SnCl.sub.2. This solution was then added to the lyophilized protein to allow Tc-99m to exchange from the phosphate groups to the sulfhydryl groups.
In our hands, the Rhodes et al. and the Schwartz et al. methods result in a labeling efficiency of about 65% and 44%, respectively. Both methods also require further purification to eliminate unbound Tc-99m. These methods, therefore, are appealing and improvement over bifunctional chelating agent methods. In a routine radiopharmacy setting, however, these methods are less than ideal because of the required purification steps. Recently Pak et al., A Rapid and Efficient Method for labeling IgG Antibodies with Tc-99m and Comparison to Tc-99m FAB' Antibody Fragments, Journal of Nuclear Medicine, Vol. 30, p. 793 (1989) and Del Rosario et al., Site-Specific Radiolabeling of Monoclonal Antibodies with Biotin/Streptavidin, Nucl. Med. Biol., (1989) used DTT (dithiothreitol) to reduced Vol, 16, pp. 525-527 (1989) used DTT (dithiothreitol) to reduced antibody disulfide groups and label them with Tc-99m. Pak et al. used a commercial glucoheptonate kit which contains SnCl.sub.2 to reduce Tc-99m. DTT and its isomer DTE (dithioerrythritol) were prepared by Cleland, Dithiothreitol, A New Protective Reagent for SH Groups, Biochemistry, Vol. 3, pp. 480-482, 1966) in 1964, and used to reduce protein disulfide groups.
In addition 2-ME has a very unpleasant, strong smell of sulfur. DTT and DTE also contain sulfur but have a less intense smell. Some patients are allergic to such compounds. For this reason, unlike ascorbic acid excess of these compounds must be removed from the proteins prior to administration to a patient.
Labeling of Tc-99m to sulfhydryl groups of antibodies has been designated by Paik et al. as high affinity but low capacity binding. Neither Rhodes et al. or Schwartz et al. show any data as to how many disulfide groups, if any, are involved and how the reduction affects the structural integrity and immunoreactivity of the antibody. Relative stability of the labeled tracer with respect to other competing chelating agents was also undetermined. A recent article by Rhodes group (Hawkins et al., Resistance fo Direct Tc-99m-Protein Bond to Transchelation, Antibody, Immunoconjugates, and Radiopharmaceuticals, Vol. 3, No. 1 (1990)) show Tc-99m-s-protein bond is stable.
Jones, A.G., Orvig, C., Trop, H.S. et al., A Survey of Reducing Agents for the Synthesis of Tetraphenylarsoniumoxotechnetium (Ethanedithiolate) From Tc-99m Pertechnetate in Aqueous Solution, J. Nucl. Med. 21, 279-281, 1980, disclose that sodium dithionite in the range of pH 11-13 gives quantitative yields of the required technetium complex through the 100% reduction of Tc-99m rapidly at room temperature. He also reported that at pH 11-13, the results were consistently quantitative.
U.S. Pat. No. 4,305,922 (Rhodes) teaches labeling proteins with Tc-99m by ligand exchange using a Sephadex column (chelating agent). A mixture of Tc-solution and stannous solution are poured into the top of the column. The protein to be labeled is then added and ligand exchanged occurs as the Tc-99m ions preferentially migrate from the chelating agent to the protein. The labeled protein is then washed through the column and recovered.
U.S. Pat. No. 4,401,646 (Rhodes et al.) discloses a method and apparatus for purifying materials radiolabeled with Tc-99m by passage through a filtration column which contains a reducing agent, colloidal stannous phthalate, anti-oxidant and a particulate solid substrate capable of binding reduced Tc and of retaining insoluble Tc. Useful anti-oxidants include gentisic acid, ascorbic acid, tartaric acid, or mixtures thereof. In this case, ascorbic acid is used to reduce the radioactive Tc only.
U.S. Pat. No. 4,416,865 (Rhodes et al.) discloses urokinase, streptokinase or fibrinokinase labeled with Tc-99m, .sup.131 I or .sup.123 I for localization of thromboembolic diseases. The enzymatic protein is combined with the nuclide in a basic solution containing ferric chloride and ascorbic acid, and the pH adjusted to acidic conditions to produce a radiopharmaceutical. In this case, ascorbic acid is used to reduce the radioactive Tc only.
U.S. Pat. No. 4,424,200 (Crockford et al.) discloses methods for radiolabeling proteins with Tc-99m in a reducing environment by incubating a source of stannous ion with a protein in the presence of a buffering composition (a mixture of an alkali metal biphtalate and alkali metal tartrate) at pH 4.5-8.5 for at least 15 hours at a temperature where the protein is not denatured.
U.S. Pat. No. 4,478,815 (Burchiel et al.) discloses a composition and method for detecting cancer with technetium labeled antibody fragments. The composition is an F(ab').sub.2 or F(ab) Fragment of antibody to hCG, hCG-.alpha., hCG-.beta. or hCG-like material or other tumor specific or associated molecules including CEA, AFP, human melanoma associated antigens labeled with Tc-99m. The radiolabeled fragment is injected intravenously, accumulates at tumor sites, and is detected by scintigraphy. A double antibody approach is also taught wherein a tumor specific antibody in the form of IgG, F(ab').sub.2 or F(ab) is administered to a patient intravenously, followed by administration of a radiolabeled antibody in the form of F(ab').sub.2 or F(ab) which is reactive with the first antibody. The radiolabeled reagents are formed under reducing conditions to minimize or prevent the reversible reaction by which the Tc-99m becomes free of the antibody fragment. The Tc-99m labeled antibody fragment may be prepared by acidic, basic or neutral (ligand exchange) radiolabeling techniques.
U.S. Pat. No. 4,652,400 (Paik et al.) discloses a direct labeling method comprising reacting a reduced radioisotope of rhenium or technetium with an antibody in the presence of diethylebnetriaminepentaacetic acid (DTPA). The DTPA inhibits binding of the radioisotopes to nonstable binding sites.
U.S. Pat. No. 4,472,371 (Burchiel et al.) discloses the labeling of radiolabeling antibodies with technetium. The Tc is reduced by stannous ions prior to labeling and the protein is also reduced using stannous chloride or stannous fluoride.
U.S. Pat. No. 4,645,660 (Takahashi et al.) discloses the reduction of a radioisotope carrier with ascorbic acid in order to increase its chelating capacity. In this case, ascorbic acid is used to reduce the radioactive Tc only.
U.S. Pat. No. 4,837,003 (Nicolotti) discloses a labeling process utilizing a coupling agent that complexes with a radionucleotide. The coupling agent is covalently bound to the Ab through sulfhydryl groups produced by mild reduction of the disulfide linkages. Reducing agents which are disclosed include 2-mercaptoethanol and, as the preferred agent, cysteine.
U.S. Pat. No. 4,851,515 (Bonnyman et al.) discloses a labeling process using the nitrodotetrahalotechnetium-99m anion and at least partial reduction of the disulfide linkages to sulfhydryl residues by use of a reducing agent such as dithiothreitol (DTT).